JP2002544476A - Multiplexed fluorescence detection in microfluidic devices - Google Patents

Abstract

(57) Abstract: An optical detection and orientation device is provided that includes a housing having an external excitation light source. An optical element for reflecting the excitation light to the aspherical lens and transmitting the light emitted by the fluorophores excited by the excitation light; an optical fiber for emitting the emitted light as a confocal aperture. A collection lens for collecting light to the entrance, and means for accurately moving the housing over a small area relative to the channel of the microfluidic device. This optical detection and orientation device finds application in the identification of the center of the channel and detection of the fluorophore of the channel during operations involving a fluorescent signal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The field of the invention is fluorescence detection in microfluidic arrays.

BACKGROUND Combinatorial chemistry, sequencing of the genomes of many species, and genotypes,
The combination of the relationship between physical and biological trials has greatly expanded the need to perform different event decisions. The diversity of novel compounds, which can be prepared using various forms of combinatorial chemistry, and the large number of targets, including wild-type and mutant genes, has led to the determination of objectives in developing compounds with biological activity. Greatly increased the number. These compounds include drugs, insecticides, pesticide resistance, disease biological resistance, and the like. Furthermore, in distinguishing differences between different genomes, in associating specific mutations with phenotypes, susceptibility to various environmental effects can be attributed to single nucleotide polymorphism.
) And in identifying the genome of an organism and providing better protection against that organism are of interest for fast and inexpensive devices and methodologies to carry out these and other decisions. Was expanded.

[0003] In recent years, microfluidic arrays have been developed that enable associating a wide variety of reservoirs and channels with small cards or chips, where various operations can be performed by using high voltages. The array provides individual networks, which are present in combination on a single chip, so that multiple decisions can be made simultaneously and / or sequentially. About 500-5000μ ²
By having a channel with a cross-section in the range, operations can be performed with very small volumes. In addition, by having a very sensitive detection system, very low concentrations of the detectable label can be used. This allows the use of very small amounts of sample and small amounts of reagents, which have become increasingly precise and expensive. Microfluidic arrays offer faster throughput, progressively shorter decision times, and the prospect of increasingly smaller amounts of required samples and reagents.

[0004] However, the use of microfluidic arrays is not without its challenges. The microfluidic array is desirably made in molded plastic to provide a reduction in chip cost. By shaping the chips and providing ridges to form the channels on the mold, the channels may not extend exactly and may deviate from their proper position, and rather than be completely straight, Can be slightly curved. In addition, plastics are often autofluorescent. Since the frequently used label is a fluorescent label, the signal from the label must be able to be distinguished from the autofluorescent signal. There is a problem with how to obtain a reliable fluorescent signal, ie, maximizing the signal from the detectable label while minimizing the background signal.

In addition, the channel walls are not orthogonal to the cover plate, so that the irradiation depth varies depending on where the excitation beam enters the channel. At the location where the excitation beam encounters the wall, the signal degrades due to the reduced number of excited fluorophores and the excitation of the wall fluorophores. Therefore, accurate positioning of the excitation beam in the channel is necessary for reproducible and accurate results.

BRIEF DESCRIPTION OF RELATED ART A number of patents have been published describing systems for detecting fluorescent signals in capillary arrays. For example, U.S. Patent Nos. 5,296,703 and 5,730,850, and WO 98/49543.

SUMMARY OF THE INVENTION An optical fluorescence detection system is provided for use with a microfluidic array. The detection and orientation system includes an optical train for receiving and processing light from a light source and directing the light onto a microfluidic channel of a solid substrate.
This optical train is moved across the surface of the solid substrate, traversing the channel,
Then, light emitted from the solid substrate is received. The optical train directs and processes light from the solid substrate surface, and directs the light to a detector.
The signal from the detector was received by a data analyzer, which analyzed the signal and observed an optical train from the bulk material of the solid substrate, the edge of the channel, and the channel. In relation to the signal, it points towards the center of this channel. The fluorescent component in the channel is detected by the fluorescence generated by the excitation light, wherein the emitted light is processed by an optical train and analyzed for the presence of fluorescence in the channel resulting from the fluorescent component in the channel;
Correct any fluorescence from the solid substrate.

The optical fluorescence detection system uses a plurality of miniature confocal microscope systems that are aligned and aligned with a plurality of channels of a microfluidic array. The system is mounted on a movable support for alignment with a set of channels. The support may be mounted on a carriage for alignment with different sets of channels. The illumination unit comprises a light source and processing means for removing light outside the desired wavelength range (e.g.,
Lenses, dichroic mirrors, filters, gratings, etc.). A single light source may be used, and the beam splits into multiple optical fibers for individual distribution of the beamlets for channel illumination. Similarly, individual signals from each channel are directed by individual optical fibers to a conventional detector. Alternatively, a separate light source (eg, an LED or laser diode) may be used for each confocal microscope system.

[0009] This methodology allows for accurate and reproducible determination of the fluorescent signal from each of the channels. To achieve the desired detection sensitivity, the center of each channel is determined, either when the channels are empty (air) or when a liquid (usually containing a fluorescent dye) is present. Depending on the degree of autofluorescence of the microfluidic array substrate, this optical system can fluoresce when there is sufficient autofluorescence to provide a detectable signal or scattered light, usually when autofluorescence is low. Can be inspected. In the case of scattered light, a wavelength different from light generated from autofluorescence is detected.

[0010] There are two different forms of excitation delivery: single mode fiber delivery or without fiber, where laser and splitting must be done by separate mirrors or diffractive optics; or Multi-mode fiber delivery, where either a lamp or a laser can be used, and the splitting is accomplished by homogenizing the laser or lamp light and then splitting using a multi-mode fiber array. Done. The light source is typically a laser, which generally produces a light beam having a wavelength in the range of about 250-800 nm, usually 488 nm, 532 nm or 633 nm.

[0011] Depending on the light source, such as a laser, a filter may be used to attenuate the light intensity and minimize photobleaching and photolysis of the fluorescent label. The light is then split into multiple light beams or beamlets by a diffractive optical element, a combination of beam splitter elements (eg, a discrete mirror), or other means (eg, a discrete beam splitter and a fiber optic array). Each of the resulting beams is then directed to an individual confocal microscope associated with the channel. Either single-mode or multi-mode fibers can be used, where the multi-mode fiber optic array is used to split the illumination into N beamlets, where N is the optical to be illuminated. The number of trains). This fiber generally has a diameter in the range of about 25-75 μm, especially about 50 μm, and a length in the range of about 1-1000 mm.

[0012] The confocal housing can be very small, where the portion that houses the optical train is typically 200 mm, in combination with other housing areas typically associated with optical fibers and fixtures for the orientation system. It has a total volume of about 0.5-4 × 10 ⁴ mm ³ , with a cross section in the range of 2000 mm ² and a height in the range of about 25-200 mm. Each confocal microscope housing receives an individual light source optical fiber, such that the output surface is perpendicular to the optical axis of the housing and that the outgoing light coincides with this optical axis. , Oriented. An optical system, typically comprising a collimating lens and an objective lens, is positioned such that they focus light from the fiber into small points. These lenses are typically aspherical with a single element. They are designed to be small and still provide diffraction limited performance.

Instead of positioning the optical fiber on the optical axis, the chief ray from the optical fiber is
Outside the optical axis, it can be directed through a collimating lens that collimates the light and directs the light to a dichroic mirror. The dichroic mirror directs the chief ray along the optical axis of the housing. This chief ray is focused by a lens having a high numerical aperture (typically in the range of about 0.25 to 0.75). Irradiation spot size is about 6-10μ
m, while the collection area is about 200-600 μm ² . The excitation light excites the fluorophores present in the channel at the detection site, and the fluorescence emitted from the channel is collected by a high numerical aperture lens. If a collimating lens is used, light is directed through the collimating lens. With proper positioning and design of the collimating lens, photon losses due to obscuration by the collimating lens are minimized. If a dichroic mirror is used, it is substantially transparent over the wavelength range of interest, and the light beam collected by the collection lens is
It passes through this dichroic mirror. After passing through a dichroic mirror or collimating lens,
The light beam is usually filtered to remove light outside the wavelength range of interest,
Then, the light is re-focused on a plane including the entrance opening or the core of the multimode optical fiber. The light emitting fiber has substantially the same dimensions as the excitation fiber. The aperture acts as a confocal aperture for the confocal assembly, but there are other ways to provide a confocal pinhole (eg, avalanche photodiodes and other detectors). The emission beam is received by an emission optical fiber and directed to a detector. Various detectors with appropriate sensitivity (eg, photomultiplier tubes (PMT), charge-coupled devices (CCD), avalanche photodiodes, etc.) can be used. The signal can then be processed to provide the level of luminescence obtained from the channel, and this intensity can be correlated with the amount of fluorophore in the channel. This can serve to identify the nature of the sample, since the amount of fluorophore is related to the event of interest.

[0014] In some situations, one is interested in signals having different wavelength ranges from different fluorophores. The emitted light beam can be split into a number of different wavelengths of interest using filters, dichroic mirrors, prisms, and the like. Various commercially available systems (eg, prisms, beam splitter mirrors, etc.) are available for this purpose. This assembly with fibers preserves the mode and profile of the laser light source and ensures optimal focusing of the light beam onto the sample by the confocal microscope assembly.

The housings can be used individually, but are usually used in combination to read multiple channels at the detection site. The individual housings are mounted on a support, which is usually movable, which allows the support to move and reorient the housing relative to a different set of channels. For example, with eight housings, eight channels can be read and by being able to move the support, a different group of eight channels can be read, so that with twelve readings, 96 readings can be made. The sample from the assay plate pattern can be read. By having 12 or more housings (usually about 96 or less housings), large numbers of samples can be read quickly. This is because individual readings are made in less than a few seconds, and support movement is automated, and the entire set of readings is performed in less than about 1 minute. This support allows for movement of the housing, thereby directing the beam substantially in the center of the channel. Various methods (mechanical, electromechanical, electromagnetic, etc.) may be used to control the movement of the housing. A different method may include securing the housing to an arm mounted on a pivot rod, where the arm is constrained in one direction and pressed in the opposite direction, with the voice coil having a lever arm extending to the center of the coil. Actuator. By using a cam operated control rocker arm, or a movable support that moves in a plane, the housing can move from the midpoint to around a distance of about 10-1000μ, typically # 500μ. If the bulk material of the microfluidic chip is autofluorescent, the presence of the channel is determined by moving the illumination through a predetermined distance and detecting autofluorescence. If the bulk material is not significantly autofluorescent, then use both autofluorescence and light scatter to determine the channel signal as shown in FIG. 9 (showing the change in autofluorescence signal while illumination traverses the channel). Exists.

The control arm is rigidly connected to the housing. This control arm is pivotally mounted on the bearing so that it can move in small arcs around the channel. The arm can be actuated to scan the surface of the microfluidic chip around this arc using an optical system for fluorescence detection to determine the location of the channel. Various actuators can be used to move the arm and housing, where the movement can be accelerated and decelerated as it passes through the arc. The observed autofluorescence is transmitted to the detector and the signal is analyzed to determine the location of the channel. Once the boundaries of the channel are determined, the housing and its optical axis may be oriented substantially above the center of the channel.

The length of the housing and lever arm can be relatively short and generally ranges from 50 to 150 when measured from the axis of the bearing to the lens at the end of the housing adjacent to the microfluidic device. The movement of the housing is controlled in steps of about 0.01μ, usually in the range of about 0.1-10μ. Instead of using a mechanical arm, various electromagnetic assemblies may be used to control the movement of the housing in relation to the optical signal. If the electromagnet flux is controlled by a computer by having the opposite electromagnet, or a single electromagnet with the opposite force, this will signal the position of the housing as the housing moves through the channel area. Correlate with change. Alternatively, a motor and guide shaft may be used to move the housing, thereby allowing the housing to traverse the channel region in a plane parallel to the surface of the chip.

Preferably, a single light source is used for multiple optical systems. Light from a single light source is directed to a beam splitter, such as a system of diffractive optics or a beam splitter. Each of the beamlets is directed to an optical fiber, which transmits the light to an optical system. The light can be split into any number of rays, but usually the total number of rays does not exceed 96, usually does not exceed 64, more usually does not exceed 32, and can be as little as 4, preferably It is about 8 to 24. Each can be separated into a linear array at an angle θ, but a two-dimensional array can also be formed with the appropriate angles between the rays. Each ray has similar propagation parameters as the input beam. In particular, the divergence and transmission intensity profiles are preserved. If the transmission intensity profile of the light source is "Gaussian" or TEM ₀₀ , each ray will preserve this profile. This profile allows for optimal light collection. Each ray is propagated a sufficient distance to provide separation and different locations. This distance is generally at least 1 mm, usually about 1 to 1
, 000 mm. Individual lenses, such as aspheric lenses, achromatic doublets, etc., focus each light beam on a single mode optical fiber. Each fiber is connected to one of the confocal microscope assemblies associated with each channel.

The microfluidic array is on a solid substrate, which may be a non-flexible substrate or a flexible substrate (eg, a film). See, for example, US Pat. No. 5,750,015 for examples of microfluidic devices. When flexible, it is usually supported by and rigidly oriented with the rigid support. The channel containing the detection site generally has a depth of about 10-200 μm and a width at the channel opening in the range of about 1-500 μm, usually 10-200 μm. These channels can be parallel or various arrays, where the inlet ports can be oriented for 96 or more microtiter well plates, so that samples from the wells are And can be introduced directly into the microfluidic network. Depending on the purpose of the chip and the pattern of the channel (whether the channel is straight, curved or bent), the chip may be only 1 cm or 2 cm long, or 50 cm long, typically about 2-20 cm long , Often 12.8 cm long. The width varies with the number and pattern of the channels, and is generally at least about 1 cm, more usually at least about 2 cm, and can be 50 cm wide, often about 8.5 cm wide. The chip has an inlet port and an outlet port and usually has reservoirs for buffers and waste fluids, which are connected to the channels and connect to the main channel to move samples, reagents, etc. to the main channel There may be additional channels to be performed. Electrodes are provided in the channels, where these electrodes may be part of the chip, painted with a conductive paint, may be metal plating on the chip, or the electrodes may be external devices. To be introduced into the reservoir or channel. The spacing between the channels at the detection site is usually at least about 0.5 mm, more usually at least about 1 mm. Since these channels can take many paths and shapes, the distance between two adjacent channels can vary.

In order to make a series of decisions on a chip, the chip is introduced into a module or a group of modules, which module comprises a movable support. The tip is indexed relative to the support so that these channels are substantially oriented with respect to the optical axis of the associated housing. The module may also include electrodes or connectors to the electrodes (these are part of the chip), containers or other instruments (eg, syringes, capillaries, etc.) (these may act as sources of reagents, samples, etc. , Providing fluid movement through the ports of the chip), electrical connections between the fluorescence detector and the data analysis system, and the like. The various modules are combined so that they receive the chip and orient the chip with respect to the various components that interact with the chip. The assignment can be provided in a chip, so that it is locked in place with respect to the module and the support. Before starting operation of the channel, the housing is oriented with respect to the center of the channel. Each of the housings individually moves across the plane of the microfluidic chip, intersecting the channel at the detection zone. Depending on the level of autofluorescence in the composition of the substrate, autofluorescence or scattered light can be read. If significant autofluorescence is present, autofluorescence or scattered light can be detected and read. If the autofluorescence signal is low, the scattered light is read.

If scattered light is detected, this scatter is compared to the scatter from the channel,
Different at the edge of the channel. By observing the change in scattered light, as the housing moves across the plane of the microfluidic chip, one can detect movement from the edge of the channel to the channel, and assuming that they are equally spaced from these edges.
You can select the center.

Once the housing is secured by the designated location of the channel, the orientation process with respect to the channel and the optical housing does not necessarily have to be repeated and multiple readings can be taken. Various operations can then be performed, wherein the fluorescent label is carried to the detection site. Detection of the fluorophore label can be the result of a competition assay, nucleic acid sequencing, immunoassay, and the like.

DESCRIPTION OF SPECIFIC EMBODIMENTS For a better understanding of the present invention, consider the drawings herein. FIG. 1 shows a detection station 100. Along with this detection station, the microfluidic chip 102 is held in place by a quartz plate 104. This quartz plate may be part of a vacuum chuck (not shown), which holds the microfluidic chip 102 at a designated location fixed relative to the detection station 100. Other modes of keeping the microfluidic chip in place include gravity, force pins, pressure, clips, reversible bonding, and the like. Also shown is an electrodeized lid 106 having an electrode 108, wherein the electrode 108 is used to operate the microfluidic chip 1 during operation of the electrokinetic process.
02 port. As described above, the microfluidic chip 102 has a plurality of channels, and the system is shown here for only one channel. The detection station has an optical housing 110, which is a small tubular housing, which may be made of any convenient material (eg, plastic, aluminum, steel, etc.), and desirably It has the minimum dimensions required to accommodate the various components. The optical system uses optical elements that have been miniaturized to an acceptable degree (eg, a diffractive optical element DOE). A single DOE can serve multiple functions, for example, acting as a lens, mirror and / or grating, where this component is about 3 mm x 3 mm. The optical system includes an aspheric lens 112 at one end of the housing, juxtaposed with a channel of a microfluidic chip, which, after appropriate orientation, excites the excitation beam, as described below. Point to the center of the channel. The excitation light beam 114 is
An optical fiber connected to arm 120 of housing 110 by coupler 122 directs to dichroic mirror 116 or an equivalent optical element. Light beam 114 passes through lens 124, which acts to collect divergent light from the fiber. The excitation beam 114 is then reflected by a dichroic mirror 116, which reflects light at the excitation wavelength of interest, and light outside this reflection wavelength may pass through the dichroic mirror. The inner wall and all support elements are desirably black, thus maximizing scattered light absorption. Reflected light beam 126
Are collected by the aspheric lens 112 and form a sharp, small beam that passes through the support plate 104 to the channel 128. If a fluorophore is present in channel 128, the fluorophore is excited and emits light, which exits channel 128 and is collected by aspheric lens 112. The emitted beam passes through a dichroic mirror 116, a filter 132, where light outside the desired wavelength range is removed, and passes through a lens 134, which directs the light beam 130 to the entrance of the collection optical fiber 132. And condensing. The optical fiber is attached to the housing 110 by a coupler. Collection optical fiber 132 transmits photons to a detector (not shown).

The housing 110 is fixed to the orientation device 136 by a flange 138. Flange 138 includes housing 110, arm 120, and lever 140.
And are connected together as a movable unit. The lever 140 is rotatably mounted on a bearing 142, which is supported by a shaft 144. The orienting device 136 comprises a tubular casing 146, which is an L-bar 1
By 50, it is fixedly attached to the encoder unit 148. The casing 146 and the motor unit 148 are kept in a fixed relationship so that the movement of the lever arm 140 can be precisely controlled and the lever arm 14
The position of the zero and, in this manner, the position of the housing 110 are easily determined. The encoder 148 is connected to a rod 154 by a connector 152, on which a cam 156 is fixedly installed. The rod 154 has a bearing 158
And 160, which are placed in the tubular casing 146, thereby keeping the rod 154 in place and allowing the cam 156 to rotate from a fixed axis of rotation. Tubular housing 146 has fins 162 to which one end of a spring 164 is attached, the other end of which is attached to lever arm 140. Spring 164 restrains lever arm 140 and dashed line 16
6, the arm 140 is pressed in the direction of the fin 162 or in a counterclockwise direction. Bar 168 is supported by bushings 170 and 172, and its length provides a tight fit between cam 156 and the location of contact with lever arm 140. Thus, the distance between the surface of the cam 156 where the bar 168 is located and the lever arm 140 remains constant. As the cam 156 rotates, the bar 168 expands and contracts relative to the rod 154 on which the cam is journaled. As the lever arm 140 responds to the movement of the bar 168, the optical system within the housing 110 scans the surface for emitting fluorescent light.
As indicated above, at the boundary of the channel 128 in the microfluidic chip 102,
There is a significant reduction. Knowing the location of these boundaries and the distance between these boundaries, the encoder can be controlled to move the bar 168 to center the housing 110 on the center of the channel 128. Once the housing is centered with the channel, a motorized determination is made and the change in fluorescence monitored in channel 128, along with the change in signal resulting from the change in fluorescence intensity, is collected and collected by collection fiber 132. Directed to an analysis device (not shown).

The microfluidic chip can be oriented to have a single channel within the boundaries of a single housing width, so that the determination of the center of the channel is orthogonal to that channel. Alternatively, the channel may be angled with respect to the path of the housing, so that the measurement is angled with respect to the channel boundary and still allows the center to be determined. Instead of having the housing as rows, the housings can be organized in any manner that allows the boundaries of the channel to be determined at the detection site, e.g., equally spaced with arcs, multiple columns and rows Other patterns with respect to the pattern of the array or the detection site of the channel to be monitored.

FIG. 2 shows a device similar to the device shown in FIG. 1 except that there are two complete units facing each other monitoring two different channels. In this arrangement, there are two rows of devices. Since all parts are the same, the same numbers are used to indicate different components. Two detection stations 100
a and 100b adapt to each other on channels 128a and 128b. Each of the detection stations 100a and 100b has its own orientation device 136a and 136b and moves independently of each other. Having two sets of optical detection stations doubles the number of readings that can be performed simultaneously. Where the channels are properly oriented, two rows of optical detection stations monitor the two sets of channels and provide data more quickly.

In FIG. 3, a modified structure is provided, which can be used in two ways: in the first, allowing the identification of fluorophores having different absorption wavelengths; and in the second The format uses a single wavelength, but uses a different path for detection of scatter from the microfluidic chip. This figure also provides different mechanical structures for the orientation device. In the optical detection device 300, the microfluidic chip 30
2 is held in place on a vacuum chuck 306 by a glass plate 304. The microfluidic chip 302 is held at a designated position fixed with respect to the detection station 300. A lid with electrodes or other electrode source (not shown) is provided for the voltage across the channels of the microfluidic chip 302. The detection station has an optical station 310, which is a small tubular housing, which has an outer diameter of at least about 3mm, more usually at least 5mm, and usually no more than about 15mm. Diameter, more usually about 10 mm or less in outer diameter. Preferably,
The center-to-center spacing of the rowed housings is about 6-12 mm, more specifically 8-1.
0 mm. The housing can be made of any convenient material (metal or plastic) that has the smallest dimensions to accommodate the optical train and provides the desired specifications. Optical systems use optical elements that have been miniaturized to an acceptable degree (eg, diffractive optical elements). The optical system includes a microfluidic chip 30.
An aspheric lens 312 is provided at one end of the housing juxtaposed to the channel 314 in 2. Aspheric lens 312 directs the excitation beam to the center of the channel after appropriate orientation. This lens also acts to transmit a small light beam for detection of the channel 314 boundary. The housing has two dichroic mirrors, an upper dichroic mirror 316 and a lower dichroic mirror 318. These two mirrors find use because they use two different wavelengths for fluorophore excitation.
The upper excitation light beam 320 is directed by an optical fiber 322 connected to the housing 310 by a coupler 324 to an upper dichroic mirror 316 or equivalent optical element. Light beam 320 passes through a bandpass filter 326, which filters out light outside the first desired wavelength range. The excitation light beam 320 is then reflected by a dichroic mirror 316, which reflects light within the desired wavelength of light and passes the desired wavelength of emitted light. The inner walls and support elements are desirably black. The reflected light beam 328 is collected by an aspheric lens 312 to form a sharp, small beam (preferably about 5-25 μm).
m range). The illumination beam excites the fluorophores in the channel at the detection site and light is emitted. By having a beam of about 10 μm diameter,
With a channel about 50 μm wide and 100 μm deep, the irradiated volume is about ***. For a fluorophore at 50 pM concentration, the number of irradiated molecules is **
*. The emitted light passes through dichroic mirrors 318 and 316 and passes through filter 330, which filters out light outside the two different fluorophore wavelengths, and the light is coupled by an objective lens to a coupler. Is focused on the entrance of the collection optical fiber attached to the housing 310. The entrance of the collection optical fiber 334 acts as a confocal aperture. In a similar manner, the lower optical fiber 340
Is connected to the housing 310 via a coupler 342 and the light beam 32
A light beam 344 having a wavelength different from zero is directed and passes through a band-pass filter 346. Light beam 344 acts similarly to light beam 320 and is reflected by dichroic mirror 318 to channel 314, where fluorescence is emitted and aspheric lens 31
2 and collected and directed through both dichroic mirrors 318 and 316 to the confocal aperture provided by the entrance to the multimode optical fiber 334.

An orientation mechanism 348 is provided to determine the center of the channel 314, which is substantially the same as the orientation mechanism of FIG. Housing 310 is secured to orientation device 348 by bolts 350 and 352. These bolts extend through lever arm 354. In this manner, the housing 310 is fixed and connected together as a movable unit of the housing 310 and the lever 354. Lever 354 is rotatably mounted on bearing 356, which is supported by shaft 358. The orienting device 348 includes the tubular casing 36
0, and the casing is provided by an L-shaped bar 364 and a flange 366.
It is fixedly attached to the encoder unit 362. The casing 360 and the encoder unit 368 are kept in a fixed relationship so that the movement of the lever arm 354 can be precisely controlled and the position of the lever arm 354,
And in this manner, the position of the housing 310 can be easily determined. The encoder 368 is connected to a rod 372 by a connector 370, on which a cam 374 is fixedly mounted. Rod 372 passes through bearings 376 and 378, which are secured to flange 366, thereby keeping rod 372 in place and allowing cam 374 to rotate from a fixed axis of rotation. I do. The lever arm 354 has a pin 380 to which a spring 382 is attached, where the other end of the spring 382 is connected to an L-shaped bar 364.
Is fixed to a hook 384 attached to the. The spring 382 is connected to the lever arm 35
4 and press the arm 354 in the direction of the L-bar. A bar 384 is supported by bushings 386 and 388, and the length of the bar provides a tight fit between cam 374 and the contact position of lever arm 354. Therefore, the distance between the surface of the cam 374 where the bar 384 is located and the lever arm 354 is kept constant. As the cam 374 rotates, the bar 384 expands and contracts relative to the rod 372 on which the cam is journaled. Lever arm 3
As 54 responds to the movement of bar 384, the optical system in housing 310 scans the surface for emitted fluorescent light. As indicated above, there is a significant reduction at the boundaries of the channels 314 of the microfluidic chip 302. Knowing the location of these boundaries and the distance between these boundaries, the encoder can be controlled to move the bar 384 to center the housing 310 with the center of the channel 314. Once the housing is centered with the channel,
The change in fluorescence for which a motorized determination is made and which is monitored in channel 314, along with the change in signal resulting from the change in fluorescence intensity, can be obtained with collection fiber 33.
4 directed to a data collection and analysis device (not shown).

In a second use of the device, optical fiber 340 provides excitation light,
This is reflected back to the microfluidic chip 302. Dichroic mirror 316 collects the scattered light and transmits this light to collection optical fiber 322. Both dichroic mirrors 316 and 318 are transparent to the fluorescent signal emitted from channel 314, and this fluorescence is transmitted to optical fiber 334 for processing by a data processor.

In the following series of figures, common elements will not be repeated for these figures. These provide an environment for different devices for the movement of the housing and identify the central part of the channel.

In FIG. 4, the device 400 is associated with a microfluidic chip 402 and has an optical station 404, which comprises the same optics as described in FIG. Optical station 404 is secured to arm 406 by securing screws 408 and 410. The arm 406 has a bearing 412, which is mounted on a pivot rod 414. Arm 406 terminates in an electrical coil 416, which has leads 418 and 420. A magnetic bar 422 extends through the coil 420. These leads are connected to a DC current source (not shown), which is controlled by a data analyzer (also not shown). The signal from the optical system 404 is sent to a data analyzer, which detects changes in the signal as the housing traverses the plate of the microfluidic chip 402 and identifies the center of the channel. The data analyzer changes the current in the coil to move the arm 406 and scans the surface of the microfluidic chip 402. Once the center of the channel has been identified, the data analyzer fixes the position of the housing and directs the excitation light toward the center of the channel.

In FIG. 5, an alternative electromagnetic device is used. The device 500 is associated with a microfluidic chip 502 and has an optical station 504 comprising the same optics as described in FIG. An electromagnetic actuator 506 is rigidly fixed to the support 508 and faces the iron surface 510. Housing 504 is attached to flexible pivot arm 518 by bolts 514 and 516 at flange 512, which is secured to support 520 by bar 522. When the electromagnetic actuator 506 is actuated by applying a current to the electromagnetic actuator 506, a field is created that attracts the iron surface 510 to the electromagnetic actuator 506. Flexible pivot arm 518 bends and provides motive force for movement of housing 504 to electromagnetic actuator 506. Electromagnetic actuator 5
06 by changing the magnetic flux of the microfluidic chip 5.
It moves in an arc across the plane of 02 and allows detection of the center of the channel as a result of signal changes caused by light emitted from the channel. Position resolver 524 faces surface 526, where position resolver 524 detects the position of housing 504. Position resolver 524 may use sound or light to determine the distance between the position resolver and surface 526. Once the center of the channel is determined by the data analyzer, the housing 5 directing light to the center of the channel
The signal from the position resolver 524 for the position of 04 can be recorded, and the housing 504 is returned to the position for each decision in that channel. In this manner, it is not necessary to scan the surface each time one wishes to determine, but may rely on signals from the position resolver 524 to determine when the housing is properly positioned.

In the next two figures, the housing is mounted on a carrier, which is
It moves in a plane parallel to the surface of the microfluidic chip, so that light coming from the housing is always in the same direction to the microfluidic chip.

In FIG. 6, the device 600 has a microfluidic chip 601 under an optical system 602 mounted on a movable carrier 604. The movable carrier 604 is mounted on a stand 606, which has two facing support posts 608 and 610. Movement of the movable carrier 604 is controlled by a lead screw 612 that passes through a threaded channel in the movable carrier and is rotated by a motor 614 connected to the lead screw 612 by a coupler 616. Lead screw 612 is supported within post 608 by bearing 618. The two guide shafts 620 and 622 connect the posts 608 and 610
And pass through the smooth channel of the movable carrier 604 to keep the movement of the movable carrier 604 in the same plane. Motor 614 is controlled by a data analyzer, which controls the movement of movable carrier 604 and receives signals from optical system 602. Once the center of the channel has been determined, the movement of the movable carrier is stopped and maintained at the same position.

In FIG. 7, device 700 uses an electromagnetic actuator to control the movement of the optical system. One or more linear guides, such as guide shafts, guide bearings, etc., are used to maintain the optical system in a linear plane parallel to the surface of the microfluidic chip. The device 700 has a microfluidic chip 702 and an optical system 704 that includes a movable carrier 706.
Installed in As in FIG. 6, the movable carrier 706 is guided by guide shafts 708 and 710, which are
2 and 714, passing through the smooth channel of the movable carrier 706 and maintaining movement of the movable carrier 706 in a plane parallel to the upper surface of the microfluidic chip 702. The post 714 is provided with an electromagnetic actuator 716. A bar magnet 718 is provided on the side of the movable carrier 706 facing the electromagnetic actuator 716. By changing the field strength and polarity of the electromagnetic actuator 716, the movable carrier 706 allows the guide shafts 708 and 7
Along 10 can move back and forth. A detection rod 720 is attached to one of the ends of movable carrier 706 and extends through post 714 and position resolver 722. The position of the detection rod 720 is coded, for example, by gradually changing color, transmittance, reflectivity, and the like, so that the position of the detection rod 720 in the position resolver 722 can be accurately determined. Once the detection rod 72
Once a suitable position of zero has been determined, the movable carrier 706 can always be returned to the same site for further monitoring of the channels of the microfluidic chip 702. The signal from the optical system 704 is sent to a data analyzer, which also monitors the position of the detection rod 720, so that when the optical system is located in the center of the channel, the center of the microfluidic chip channel May be correlated to the position of the detection rod 720.

As indicated above, the channels may exhibit many patterns of microfluidic chips. FIG. 8 is a schematic top view of the surface of the microfluidic chip 800. The plurality of channel networks 802 include a main channel 804, an intersection channel 806, a main channel 8
It has ports and reservoirs 808 and 810 for the 04 and ports and reservoirs 812 and 814 for the intersection channel 806. Channel network 802 is arcuately spaced and "X" 816 indicates the detection site of main channel 804, where the optical housing is positioned. Instead of an arc, the channel network may be distributed to define a circle, where:
The optical housing is mounted on a platform, which is
A group of housings can rotate to address different groups of channel networks.

If desired, various electrode patterns can make up a portion of the microfluidic chip, and these electrodes can be connected to a computer or other data analysis device, which can be used to drive the operation. In the meantime, it acts to control the voltage at the various electrodes. Further, the computer may serve to control the positioning of the optical detection device during operation.

As described above, the microfluidic device has a plurality of channels, where:
Depending on the number of channels, all channels may be addressed simultaneously by the same number of optical detection devices, or some of the number of channels may be addressed at any time, and the optical detection devices or microfluidic chips or both may be Moved relative to it, it may allow the optical detection device to address a plurality of different channels. For example, using a microfluidic chip with 96 channels, with each port intended to receive samples from the wells of a 96 microtiter well plate, with 8 or 12 optical detection devices in one unit , The same number of channels may be monitored. Then, after monitoring the same number of channels, the optical detection device unit and / or microfluidic chip are moved to address a different set of channels, and the procedure is repeated until all channels are monitored.

From the above results, it is clear that the present invention provides an improved manner of detecting fluorophores in microchannels. This device and method greatly improves the signal and signal-to-noise ratio, and allows for the rapid determination of large numbers of samples, so that many channels can be monitored at once. The mechanism used can be miniaturized to be compact, while addressing multiple microchannels in a small space. Various designs of the channel are compatible with this detection system.

The present invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but with reference to the teachings of the invention, depart from the spirit and scope of the appended claims. Without departing from the invention, certain changes and modifications can be made to the invention.
It will be readily apparent to one skilled in the art.

[Brief description of the drawings]

FIG. 1 is an elevational side view of an optical detection system.

FIG. 2 is an elevational side view of a pair of optical detection systems.

FIG. 3 is an elevational side view of an alternative optical detection system.

FIG. 4 uses an electromagnetic actuator to orient the optical detection system;
FIG. 6 is an elevation view of an alternative embodiment.

FIG. 5 uses an electromagnetic actuator to orient the optical detection system;
FIG. 9 is an elevation view of an alternative embodiment using a second style.

FIG. 6 is an elevational view of an alternative embodiment that uses a carrier that moves mechanically in a plane parallel to the microfluidic substrate to orient the optical detection system.

FIG. 7 is an elevational view of an alternative embodiment that uses an electromagnetic actuator to move a carrier in a plane parallel to a microfluidic substrate to orient an optical detection system.

FIG. 8 is a top view of the surface of a microfluidic chip characterized by a plurality of channel networks.

FIG. 9 is a graph of the signals observed when orienting the optical system with respect to the channel. The conditions under which this determination was performed are as follows: laser power 2 mW; 10 micron in spot size FWHM; acrylic microfluidic chip, 30 micron deep channel, HEPES buffer (50 mM, pH 7.4).
80 micron width filled with; scanning across the open channel (back and forth) about 400 microns / sec; 488 nm excitation (argon ion laser); 530 nm emission filter;
physical System; focus usually set for optimal signal performance.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Smith, Timothy United States of America 94553, Martinez, Palisade Court 1014 (72) Inventor Bhujanson, Toleif United States of America 95020, Gilroy, Daniel Court 7030 F-term (reference) 2G043 AA03 BA16 CA03 DA06 EA01 FA02 GA02 GA04 GB01 HA01 HA02 HA05 HA09 JA02 KA01 KA02 KA03 KA09 LA02 LA03

Claims (20)

[Claims] 1. An optical detection and orientation system for illuminating a fluorescent sample in a microchannel and for detecting light emitted from a solid substrate and fluorescence emitted from the channel, the system comprising: Is present on a solid substrate, the system comprising: a movable optical train, comprising: an excitation light source and means for directing the light onto the solid substrate; receiving light emitted from the solid substrate; Means for transmitting the emitted light for analysis; and moving the optical train and excitation light relative to a surface of the solid substrate with the microchannels, and changing light emitted from the solid substrate. Means for aligning the optical train with respect to the microchannel in response to the optical detection and orientation system. 2. The optical detection and orientation system according to claim 1, wherein the means for receiving the emitted light is part of the movable optical train. 3. The optical detection and orientation system according to claim 1, wherein the emitted light is fluorescent light. 4. The optical detection and orientation system according to claim 1, wherein the emitted light is scattered light. 5. An optical detection and orientation system for illuminating a fluorescent sample in a microchannel and for detecting light emitted from a solid substrate and fluorescence emitted from the channel, wherein the microchannel comprises: Resides on a solid substrate, the system comprising: a movable optical unit comprising an optical train, an excitation light source, and reflective and transmissive optical elements for directing an excitation light beam from the light source to an aspheric lens. An aspheric lens for collecting the light emitted from the microchannel and collecting the light emitted from the microchannel; and An aspheric lens that collects and directs light through the reflective and transmissive optical element, wherein the reflective and transmissive element reflects the excitation light beam; A movable optical unit that is an element that transmits the emitted light; and a condensing lens for receiving the emitted light and directing a focused beam of the emitted light to an entrance of an optical fiber. A converging lens, wherein the entrance acts as a confocal aperture; fixed to the carrier, means for accurately moving the carrier and the unit over a short distance; and from the optical fiber to the data analyzer. A connector, wherein the data analyzer analyzes changes in light emitted from the substrate as the carrier and unit move over the surface of the substrate, and relates to a pattern of fluorescence received from the optical fiber. A connector, which is an analyzer for controlling the moving means to move the housing, comprising: a connector; 6. The optical detection and orientation system according to claim 5, comprising means for orienting the substrate with the microchannels in a fixed position with respect to the housing. 7. The optical detection and orientation system according to claim 5, wherein the carrier is a rotatable lever arm. 8. The moving means for connecting and pressing a rotating cam mounted on a motor drive shaft, a bar located between the cam and the housing, and the movable optical unit to the shaft. The optical detection and orientation system according to claim 7, comprising a propulsion means. 9. The optical detection and alignment system according to claim 5, wherein the carrier is a support, and the support moves on a linear guide. 10. The optical detection and orientation system according to claim 9, wherein the support is moved by mechanical means. 11. The optical detection and orientation system according to claim 9, wherein the support is moved by electromagnetic means. 12. The optical detection and alignment system according to claim 5, wherein the optical train comprises three optical fibers and two reflective and transmissive elements, wherein the optical fibers and the reflective and transmissive elements are: In a first embodiment, for directing two different excitation light beams of different wavelengths and for receiving the two different excitation light beams,
(2) In the second embodiment, the first optical fiber for directing the excitation light to the first reflection / transmission element and the second optical fiber for receiving the light emitted from the substrate are used. Wherein the first reflective and transmissive element directs the excitation light to the substrate, and transmits light from the substrate to a second reflective and transmissive element; Receives the light emitted from the substrate and reflects the emitted light to the second optical fiber, wherein the first and second reflective and transmissive elements reduce the fluorescence emitted from the channel. An optical detection and orientation system that transmits to a third optical fiber. 13. The optical detection and orientation of claim 5, further comprising a data analysis unit for receiving signals from the emitted light and controlling the moving means to move the optical unit. system. 14. A filter between the condenser lens and the reflection / transmission element, further comprising a filter for filtering light of the radiated light outside a target wavelength range. 6. The optical detection and orientation system according to 5. 15. An optical detection and orientation system for illuminating a fluorescent sample in a microchannel and for detecting light emitted from a solid substrate and fluorescence emitted from the channel, wherein the microchannel comprises: Is present on the solid substrate,
The system comprises: a movable optical unit containing an optical train, an excitation light source, and reflective and transmissive optics for directing an excitation light beam from the light source to an aspheric lens.
A housing, and a carrier rigidly fixed to the housing, wherein the aspheric lens collects the light on the microchannel and collects light emitted from the microchannel, and transmits the emitted light. A movable unit, wherein the movable unit is an aspheric lens that condenses and directs through the reflecting and transmitting element, wherein the reflecting and transmitting element reflects the excitation light beam and transmits the emitted light. A collection lens for receiving the emitted light and directing a focused beam of emitted light to an optical fiber entrance, wherein the entrance acts as a confocal aperture; An optical lens fixed to the carrier, means for accurately moving the carrier and the unit in an arc over a short distance; and a connector from the optical fiber to a data analyzer. The data analyzer is further configured to analyze changes in light emitted from the substrate as the carrier and unit move over the surface of the substrate, and to analyze the pattern of radiation received from the optical fiber with the housing. A connector, which is an analyzer for controlling the moving means to move the light. 16. The optical detection and orientation system according to claim 15, wherein said moving means comprises a cam for controlling movement of said carrier. 17. An optical detection and orientation system for illuminating a fluorescent sample in a microchannel and for detecting light emitted from a solid substrate and fluorescence emitted from the channel, wherein the microchannel comprises: Is present on the solid substrate,
The system comprises: a movable optical unit containing an optical train, an excitation light source, and reflective and transmissive optics for directing an excitation light beam from the light source to an aspheric lens.
A housing, and a carrier rigidly fixed to the housing, wherein the aspheric lens collects the light on the microchannel and collects light emitted from the microchannel, and transmits the emitted light. A movable unit, wherein the movable unit is an aspheric lens that condenses and directs through the reflecting and transmitting element, wherein the reflecting and transmitting element reflects the excitation light beam and transmits the emitted light. A condensing lens for receiving the radiation and directing a focused beam of radiation from the substrate to an optical fiber entrance, wherein the entrance is a confocal aperture; A working condensing lens; means fixed to the carrier for accurately moving the carrier and the unit over a short distance in a plane parallel to the substrate; and A connector to a data analyzer, wherein the data analyzer analyzes changes in light emitted from the substrate as the carrier and the unit move over the surface of the substrate, and receives data from the optical fiber. A connector, which is an analyzer for controlling the moving means to move the housing with respect to the defined light pattern. 18. The moving means comprises a carrier on a guide shaft.
An optical detection and orientation system according to claim 17. 19. The optical detection and orientation system according to claim 18, wherein said moving means further comprises electromagnetic means for moving said carrier on said guide shaft. 20. The threaded shaft in a threaded sleeve in the carrier, and one end of the threaded shaft for rotating the threaded shaft to move the carrier. 18. The optical detection and orientation system of claim 17, further comprising a motor connected to the.